JP5230224B2 - New freeze-resistant baker's yeast - Google Patents
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Description
本発明は、冷凍耐性を賦与又は強化したパン酵母、及びそれに係る遺伝子並びに該冷凍耐性パン酵母を用いてなる冷凍パン生地及び冷凍生地製パン法に関する。 The present invention relates to baker's yeast imparted or enhanced freezing tolerance, a gene related thereto, a frozen bread dough using the freeze-resistant baker's yeast, and a method for producing a frozen dough.
冷凍生地製パン法とは、製パン工程の途中でパン生地を一時冷凍し、必要に応じて解凍、焼成する製パン法である。この製法では、パン生地冷凍中の輸送、貯蔵が可能であるため、主に深夜、早朝に行われる製パン作業の労働条件の改善に有効である。また、焼きたてパンの需要や、多品種少量生産にも対応できることから、広く普及している。しかしながら、生地を冷凍することにより、生地中のパン酵母は致命的な傷害を受けるため、通常の製パン法による製品の品質には及ばないという欠点がある。そのため、製品価値のあるパンを得るためには、冷凍傷害を受けにくい酵母、すなわち冷凍耐性を有する酵母が必要である。近年では、イーストメーカー各社において、様々な冷凍耐性パン酵母が開発され、機能は向上してきている。しかし、まだまだ充分ではなく、更なる機能向上のために冷凍耐性のメカニズムに関する研究が行われているが、未だ解明には至っていない。 The frozen dough bread making method is a bread making method in which bread dough is temporarily frozen during the bread making process and then thawed and baked as necessary. This manufacturing method can be transported and stored while the dough is frozen, and is therefore effective in improving the working conditions of the bread-making work performed mainly at midnight and early morning. In addition, it is widely used because it can meet demand for freshly baked bread and small-lot production of various varieties. However, when the dough is frozen, the baker's yeast in the dough is fatally damaged, so that there is a disadvantage that it does not reach the quality of the product by the usual baking method. Therefore, in order to obtain bread having product value, yeast that is not easily damaged by freezing, that is, yeast having freezing tolerance is required. In recent years, various freeze-tolerant baker's yeasts have been developed by yeast manufacturers and their functions have been improved. However, it is still not enough, and research on the mechanism of freezing tolerance has been conducted for further functional improvement, but it has not yet been elucidated.
冷凍耐性パン酵母に関する従来技術としては、トレハラーゼをコードする遺伝子ＮＴＨ１、ＡＴＨ１を不活性化（遺伝子破壊）することにより、細胞内のトレハロースを高蓄積させた酵母（特許文献１、特許文献２）、脂肪酸不飽和化酵素をコードするＯＬＥ１遺伝子の量を増加させることにより、細胞内の不飽和脂肪酸の割合を増加させた酵母（特許文献３）、アルギニン分解酵素をコードするＣＡＲ１遺伝子を不活性化することによって、特定アミノ酸を高蓄積させた酵母（特許文献４）、ＹＬＲ０２３Ｃ遺伝子及び／又はＹＭＲ１２６Ｃ遺伝子の発現量を上昇させた酵母（特許文献５）等が知られている。 As conventional techniques relating to freeze-tolerant baker's yeast, yeast in which trehalose is highly accumulated by inactivating (gene disruption) the genes NTH1 and ATH1 encoding trehalase (Patent Documents 1 and 2), By increasing the amount of OLE1 gene encoding fatty acid desaturase, yeast in which the percentage of unsaturated fatty acid in the cell is increased (Patent Document 3), CAR1 gene encoding arginine degrading enzyme is inactivated. Thus, there are known yeast (Patent Document 4) in which specific amino acids are highly accumulated, yeast (PTL 5) in which the expression level of YLR023C gene and / or YMR126C gene is increased, and the like.
上記のように、酵母の冷凍耐性には複数の遺伝子が関与することが示唆されており、上記した遺伝子以外にも幾つかの遺伝子が関与していると考えられる。 As described above, it has been suggested that a plurality of genes are involved in the freezing tolerance of yeast, and it is considered that several genes are involved in addition to the genes described above.
変異型のプロリン合成酵素を高発現させることによりプロリンを高蓄積させた酵母（非特許文献１）や、グリセロール分解酵素をコードする遺伝子を不活性化することにより、細胞内のグリセロールを高蓄積させた酵母（非特許文献２）も報告されている。 By inactivating the yeast that highly accumulated proline by highly expressing the mutant proline synthase (Non-patent Document 1) and the gene encoding glycerol-degrading enzyme, the glycerol in the cell is highly accumulated. Yeast (Non-patent Document 2) has also been reported.
通常実用パン酵母の冷凍耐性は、パン生地を冷凍保存した後でも発酵力が充分保持されている事で「冷凍耐性有り」と評価出来る。しかしながら上記の如き従来技術では、遺伝子操作が容易ではあるがパン生地でのガス発生量（所謂、発酵力）が実用レベルに無い実験室酵母を用いて冷凍耐性を検証しており、パン生地でのガス発生量を指標とせず、生菌数あるいは僅かな液系発酵を指標とする冷凍耐性試験に過ぎなかったり、また実用パン酵母を用いていても、実際の発酵力は低くなっており、これらの技術で得られた実用パン酵母の冷凍耐性は充分でなかった。従って、実際のパン生地系での冷凍耐性の強化あるいは賦与への応用が可能な知見や技術の開発が求められている。 Usually, the freezing tolerance of commercial baker's yeast can be evaluated as “with freezing tolerance” because the fermentative power is sufficiently maintained even after the dough is stored frozen. However, in the prior art as described above, the freezing tolerance is verified using laboratory yeasts that do not have a practical level of gas generation amount (so-called fermentation power) in bread dough, although genetic manipulation is easy. It is only a freezing tolerance test that does not use the generated amount as an index, but uses the number of viable bacteria or a slight amount of liquid fermentation as an index, and even if a practical baker's yeast is used, the actual fermentative power is low. The freezing tolerance of the practical baker's yeast obtained by the technique was not sufficient. Accordingly, there is a need for the development of knowledge and technology that can be applied to strengthening or imparting freezing tolerance in actual bread dough systems.
また、特に最近の技術として、冷凍耐性に関する多くの遺伝子を一度にスクリーニングすることを目的とした解析も行われている（非特許文献３）。この報告は、遺伝子破壊株ライブラリーを用いた全遺伝子網羅的な解析であり、比較的緩慢な冷凍条件（−２５℃）で冷凍耐性を評価しているため、多くの遺伝子が冷凍耐性に関わることを示している。但しこの報告においても、各遺伝子破壊菌株の冷凍耐性は、液系での冷凍後菌体生存率を指標に評価されているに過ぎず、実用冷凍耐性の改良に応用できる遺伝子のスクリーニングあるいは絞り込みができていなかった。さらには、上記遺伝子破壊株ライブラリーを用いたスクリーニングにおいて、その条件がドライアイス・エタノールバス（−８０℃）を使用するような過酷な冷凍条件ではなく、冷凍耐性への影響が大きい遺伝子、即ち実用機能レベルで有用な冷凍耐性を特には選別できていなかった。
本発明の目的は、実用パン酵母の冷凍耐性形質に関連する遺伝子を明らかにし、冷凍耐性を賦与あるいは強化したパン酵母を作製することである。さらには、冷凍耐性パン酵母の新規な作製方法を提供することである。 An object of the present invention is to elucidate genes related to freezing tolerance traits of practical baker's yeast and to produce baker's yeast imparted or enhanced freezing tolerance. Furthermore, it is providing the novel preparation method of freezing tolerance baker's yeast.
本発明者らは上記課題を解決する為に鋭意研究を重ねた結果、実用パン酵母の遺伝子としてＫＴＲ１遺伝子やＭＮＴ３遺伝子を発現させたパン酵母は、冷凍耐性が上がることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that baker's yeast expressing KTR1 gene and MNT3 gene as practical baker's yeast genes has increased freezing tolerance and completed the present invention. It came to do.
即ち、本発明の第一は、配列番号１に示すサッカロミセス・セレビシエのＫＴＲ１遺伝子又は該遺伝子の相補鎖にハイブリダイズ可能な遺伝子からなる冷凍耐性遺伝子に関する。本発明の第二は、配列番号２に示すサッカロミセス・セレビシエのＭＮＴ３遺伝子又は該遺伝子の相補鎖にハイブリダイズ可能な遺伝子からなる冷凍耐性遺伝子に関する。本発明の第三は、上記記載のＫＴＲ１遺伝子又は該遺伝子の相補鎖にハイブリダイズ可能な遺伝子からなる冷凍耐性遺伝子によりコードされ、パン酵母の発酵において冷凍耐性を賦与又は強化するタンパク質に関する。本発明の第四は、上記記載のＭＮＴ３遺伝子又は該遺伝子の相補鎖にハイブリダイズ可能な遺伝子からなる冷凍耐性遺伝子によりコードされ、パン酵母の発酵において冷凍耐性を賦与又は強化するタンパク質に関する。本発明の第五は、上記記載のＫＴＲ１遺伝子及び／又は上記記載のＭＮＴ３遺伝子を用いて形質転換させてなる、冷凍耐性を賦与または強化したパン酵母に関する。好ましい実施態様は、パン酵母が、サッカロミセス・セレビシエに属するＧＭＫＹ７９株（受託番号：ＮＩＴＥ Ｐ−４３６）である上記記載のパン酵母、或いは、パン酵母が、サッカロミセス・セレビシエに属するＧＭＫＹ８０株（受託番号：ＮＩＴＥ Ｐ−４３７）である上記記載のパン酵母、に関する。本発明の第六は、上記記載のＫＴＲ１遺伝子及び／又は上記記載のＭＮＴ３遺伝子を用いて形質転換することを特徴とするパン酵母の冷凍耐性を向上させる方法に関する。 That is, the first of the present invention relates to a freezing tolerance gene comprising the Saccharomyces cerevisiae KTR1 gene shown in SEQ ID NO: 1 or a gene hybridizable to the complementary strand of the gene. The second of the present invention relates to a freezing resistance gene comprising the MNT3 gene of Saccharomyces cerevisiae shown in SEQ ID NO: 2 or a gene capable of hybridizing to the complementary strand of the gene. The third aspect of the present invention relates to a protein that is encoded by a freeze tolerance gene comprising the KTR1 gene described above or a gene that can hybridize to the complementary strand of the gene, and that imparts or enhances the freeze tolerance in baker's yeast fermentation. The fourth aspect of the present invention relates to a protein that is encoded by a freezing tolerance gene comprising the MNT3 gene described above or a gene that can hybridize to the complementary strand of the gene, and that imparts or enhances freezing tolerance in baker's yeast fermentation. A fifth aspect of the present invention relates to a baker's yeast imparted with or enhanced freezing tolerance, which is transformed with the above-described KTR1 gene and / or the above-described MNT3 gene. In a preferred embodiment, the baker's yeast is GMKY79 strain (accession number: NITE P-436) belonging to Saccharomyces cerevisiae, or the baker's yeast is GMKY80 strain (accession number: NITE P-437). The sixth aspect of the present invention relates to a method for improving the freezing tolerance of baker's yeast, which comprises transforming with the above-described KTR1 gene and / or the above-described MNT3 gene.
本発明によれば、実用パン酵母の冷凍耐性形質に関連する遺伝子を明らかにでき、冷凍耐性を賦与あるいは強化したパン酵母の作製することが可能となる。さらには、冷凍耐性パン酵母の新規な作製方法を提供できる。 According to the present invention, genes related to the freezing tolerance traits of practical baker's yeast can be clarified, and baker's yeast imparted with or enhanced freezing tolerance can be produced. Furthermore, it is possible to provide a novel method for producing freeze-resistant baker's yeast.
以下、本発明についてさらに詳細に説明する。本発明の冷凍耐性遺伝子は、配列番号１に示すサッカロミセス・セレビシエのＫＴＲ１遺伝子又は該遺伝子の相補鎖にハイブリダイズ可能な遺伝子、或いは、配列番号２に示すサッカロミセス・セレビシエのＭＮＴ３遺伝子又は該遺伝子の相補鎖にハイブリダイズ可能な遺伝子からなり、該遺伝子は、パン酵母の発酵において冷凍耐性を賦与又は強化するタンパク質をコードする。そして、該遺伝子を用いた形質転換体であるパン酵母において、該遺伝子を高発現させることで冷凍耐性が賦与または強化されている。 Hereinafter, the present invention will be described in more detail. The freezing tolerance gene of the present invention is a gene that can hybridize to the KTR1 gene of Saccharomyces cerevisiae shown in SEQ ID NO: 1 or a complementary strand of the gene, or the MNT3 gene of Saccharomyces cerevisiae shown in SEQ ID NO: 2 or the complement of the gene. It consists of a gene that can hybridize to the chain, which encodes a protein that confers or enhances freezing tolerance in baker's yeast fermentation. In baker's yeast, which is a transformant using the gene, freezing tolerance is imparted or enhanced by high expression of the gene.
サッカロミセス・セレビシエには、約６０００個の遺伝子があり、そのうち生存にとって必須な遺伝子約１２００個を除いた４７９２個の遺伝子を１つ宛破壊した４７９２種類の遺伝子破壊株（4792 yeast deletion strains）が販売されている。本発明者らは、この実験室株由来の単一遺伝子破壊株セット（以降、遺伝子破壊株ライブラリーと呼ぶ）から、冷凍耐性遺伝子をスクリーニングした。具体的には、各遺伝子破壊菌株に対して、冷凍後のコロニー生育度合いを目視評価する一次スクリーニングと、冷凍後の生菌数を計測評価する二次スクリーニングを実施し、冷凍耐性への影響が明確な破壊株すなわち冷凍耐性遺伝子を選択した。冷凍条件は、約−８０℃にもなるドライアイス・エタノールバスで冷凍処理を施すことにより冷凍耐性への影響が大きい遺伝子のスクリーニングを行った。 Saccharomyces cerevisiae has about 6000 genes, of which 4792 types of gene-disrupted strains (4792 yeast deletion strains) are sold, with 4792 genes excluding about 1200 genes essential for survival being destroyed. Has been. The present inventors screened a freezing resistant gene from a single gene disrupted strain set derived from this laboratory strain (hereinafter referred to as gene disrupted strain library). Specifically, for each gene-disrupted strain, a primary screening that visually evaluates the degree of colony growth after freezing and a secondary screening that measures and evaluates the number of viable cells after freezing are performed, and the effect on freezing tolerance is A clear disruption strain, namely the freezing tolerance gene, was selected. As freezing conditions, a gene having a large influence on freezing tolerance was screened by performing a freezing treatment in a dry ice / ethanol bath of about −80 ° C.
これにより、遺伝子破壊により明確に冷凍感受性となる菌株があることを確認し、これまでに報告の無かった新たな冷凍耐性遺伝子を複数個スクリーニングできた。スクリーニングされた遺伝子の内、ＫＴＲ１遺伝子及びＭＮＴ３遺伝子は、タンパク質への糖鎖付加に関わるタンパク質をコードする遺伝子として報告があり（Glycobiology, 1999, Oct;9(10):1045-51）、糖鎖付加は、タンパク質の活性制御に重要な役割を果たしていることが知られていたが、前記のようなＫＴＲ１遺伝子あるいはＭＮＴ３遺伝子の破壊による冷凍耐性の低下、即ちＫＴＲ１遺伝子やＭＮＴ３遺伝子が冷凍耐性に関与することは本発明により初めて明かとなった。 As a result, it was confirmed that there was a strain that clearly became freezing-sensitive by gene disruption, and a plurality of new freezing-resistant genes that had not been reported so far could be screened. Among the screened genes, KTR1 gene and MNT3 gene have been reported as genes encoding proteins involved in glycosylation to proteins (Glycobiology, 1999, Oct; 9 (10): 1045-51). Addition was known to play an important role in protein activity control, but the above-mentioned decrease in freezing tolerance due to disruption of the KTR1 gene or MNT3 gene, that is, the KTR1 gene and MNT3 gene are involved in freezing tolerance. This was first clarified by the present invention.
従って、変異等により活性を向上させたＫｔｒ１タンパク（ＫＴＲ１遺伝子がコードするタンパク質）又はＭｎｔ３タンパク（ＭＮＴ３遺伝子がコードするタンパク質）を用いるか、或いはＫｔｒ１タンパク又はＭｎｔ３タンパクの量、即ちＫＴＲ１遺伝子又はＭＮＴ３遺伝子の発現量を増加させることで、パン酵母の冷凍耐性を賦与または強化することができる。そのためには、ＫＴＲ１遺伝子及び／又はＭＮＴ３遺伝子を用いて形質転換したり、各遺伝子のプロモーターを強化したりすることなどが好ましい。その中でも、ＫＴＲ１遺伝子及び／又はＭＮＴ３遺伝子を含むプラスミドを用いて形質転換することが操作上容易なため好ましい。 Therefore, Ktr1 protein (protein encoded by KTR1 gene) or Mnt3 protein (protein encoded by MNT3 gene) whose activity has been improved by mutation or the like is used, or the amount of Ktr1 protein or Mnt3 protein, ie, KTR1 gene or MNT3 gene By increasing the expression level, the freezing tolerance of baker's yeast can be imparted or enhanced. For that purpose, it is preferable to transform using the KTR1 gene and / or MNT3 gene, or to enhance the promoter of each gene. Of these, transformation using a plasmid containing the KTR1 gene and / or the MNT3 gene is preferable because of ease of operation.
具体的には、ＫＴＲ１遺伝子を含むプラスミドを用いた形質転換体であるＧＭＫＹ７９株が、ＮＩＴＥ Ｐ−４３６（受託番号）として独立行政法人製品評価技術基盤機構（日本国千葉県木更津市かずさ鎌足２−５−８）に寄託されている。また、ＭＮＴ３遺伝子を含むプラスミドを用いた形質転換体であるＧＭＫＹ８０株が、ＮＩＴＥ Ｐ−４３７（受託番号）として独立行政法人製品評価技術基盤機構（日本国千葉県木更津市かずさ鎌足２−５−８）に寄託されている。これらの株は、何れも発現量が形質転換前の１０倍以上に増加している。即ち、前記株を用いてパン生地を作製することで、冷凍耐性の高いパン生地が得られる。また、対照となる前記タンパク質の非高発現株としてプラスミドｐＥＵＡを用いた形質転換体であるＧＭＫＹ７８株が、ＮＩＴＥ Ｐ−４３５（受託番号）として独立行政法人製品評価技術基盤機構（日本国千葉県木更津市かずさ鎌足２−５−８）に寄託されている。 Specifically, the GMKY79 strain, which is a transformant using a plasmid containing the KTR1 gene, was designated as NITE P-436 (Accession Number) by the National Institute of Technology and Evaluation (Kazusa Kamashi 2, Kisarazu City, Chiba Prefecture, Japan). -5-8). In addition, GMKY80 strain, which is a transformant using a plasmid containing the MNT3 gene, is designated as NITE P-437 (accession number) by the National Institute of Technology and Evaluation (Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan). 8). In all of these strains, the expression level is increased 10 times or more before transformation. That is, bread dough having high freezing resistance can be obtained by producing bread dough using the strain. In addition, GMKY78 strain, which is a transformant using plasmid pEUA as a non-high expression strain of the above-mentioned protein as a control, was incorporated as NITE P-435 (accession number) by National Institute of Technology and Evaluation (Kisarazu, Chiba Prefecture, Japan). It has been deposited with Kazusa City Kamashita 2-5-8).
なお、ＫＴＲ１遺伝子及びＭＮＴ３遺伝子は、パン酵母の冷凍耐性に寄与していることが認められたが、それぞれの遺伝子の相補鎖にハイブリダイズ可能な遺伝子も、ＫＴＲ１遺伝子やＭＮＴ３遺伝子と同等以上の冷凍耐性を発現できる限り有効である。 The KTR1 gene and the MNT3 gene were found to contribute to the freezing tolerance of baker's yeast, but the genes that can hybridize to the complementary strands of the respective genes are also frozen at the same level or higher than the KTR1 gene and the MNT3 gene. It is effective as long as tolerance can be expressed.
（冷凍耐性の規定と評価方法）
冷凍耐性パン酵母とは、一般に冷凍による傷害を受けにくいパン酵母のことを言う。本発明における冷凍耐性とは、［１］ドライアイス・エタノールバスなどによる−８０℃での急速冷凍−解凍後の菌体生存率（冷凍耐性度）が、液系評価法で対照となる非破壊株と比較して２０％以上高い場合を冷凍耐性、同様に２０％以上低い場合を冷凍感受性、或いは、［２］１週間冷凍−解凍後の生地のガス残存率（冷凍耐性度）が、生地系評価法で対照となる非高発現株と比較して２０％以上高い場合を冷凍耐性、同様に２０％以上低い場合を冷凍感受性とする。ここで液系評価法とは、スクリーニングした遺伝子破壊株について、ＹＰＤ寒天培地上に生育したコロニー数をカウントすることにより冷凍後の生存率を測定する方法である。詳細には、ＹＰＤ培地に適量の菌体を植菌して一晩前培養した後、培養液を新しいＹＰＤ培地に植菌し、一晩本培養した後、培養液をマイクロチューブ２本に分注し、１本は滅菌水で適宜希釈して、ＹＰＤ寒天培地に塗布し、もう１本はドライアイス・エタノールバスで冷却後、冷凍庫で冷凍保存する。１日冷凍後、解凍した培養液サンプルを、滅菌水で適宜希釈して、ＹＰＤ寒天培地に塗布し、冷凍前及び冷凍後のサンプルを塗布した寒天培地に生育したコロニーをカウントし、冷凍後の菌体生存率（冷凍後サンプルのコロニー数／冷凍前サンプルのコロニー数）を指標として冷凍耐性の評価を行う。上記のような液系生存率による評価法では、実用機能レベルの発酵力を持たない株でも冷凍耐性を評価できるので、遺伝子破壊株ライブラリーから実用パン酵母まで、様々な菌株の冷凍耐性評価に有効に用いることができる。
(Definition of freezing tolerance and evaluation method)
The freeze-tolerant baker's yeast generally refers to baker's yeast that is not easily damaged by freezing. The freezing tolerance in the present invention is [1] non-destructive method in which the cell viability (freezing tolerance) after thawing at −80 ° C. with a dry ice / ethanol bath or the like is a control in the liquid system evaluation method. When the content is 20% or higher compared to the stock, the freezing resistance is the same, and when the content is 20% or lower, the freezing sensitivity, or [2] The gas residual rate (freezing tolerance) of the dough after freezing and thawing for 1 week The case where it is 20% or more higher than the non-high-expressing strain which is the control in the system evaluation method is the freezing tolerance, and the case where it is 20% or more lower is the freezing sensitivity. Here, the liquid system evaluation method is a method of measuring the survival rate after freezing by counting the number of colonies grown on the YPD agar medium for the screened gene disruption strain. Specifically, after inoculating an appropriate amount of cells in a YPD medium and pre-culturing overnight, the culture solution is inoculated into a new YPD medium and main culture is performed overnight, and then the culture solution is divided into two microtubes. Note, one is diluted appropriately with sterilized water and applied to the YPD agar medium, and the other is cooled in a dry ice / ethanol bath and stored frozen in a freezer. After freezing for 1 day, the thawed culture solution sample is appropriately diluted with sterilized water and applied to the YPD agar medium. The colonies grown on the agar medium coated with the sample before freezing and after freezing are counted. Evaluation of freezing resistance is performed using the cell viability (number of colonies after freezing / number of colonies before freezing) as an index. In the evaluation method based on the liquid system survival rate as described above, freezing tolerance can be evaluated even for strains that do not have fermentative ability at the level of practical function. It can be used effectively.
こういった生存率を指標とする液系冷凍耐性評価は、発酵力や増殖性が実用レベルに無い上記の如きライブラリー株を直接かつ効率的に評価し得る数少ない方法といえるが、生地系評価との発酵環境の違いは歴然としており、正確なスクリーニングのためには適正な試験条件の設定が重要である。更には、液系評価に留まること無く、より正確で実際に即した生地系発酵力評価を実施することにより実用パン酵母としての冷凍耐性が評価できる。 Although liquid freezing tolerance evaluation using such a survival rate as an index can be said to be one of the few methods that can directly and efficiently evaluate the above-mentioned library strains that do not have fermentative ability and proliferation ability at practical levels, dough-based evaluation The difference in fermentation environment is obvious, and it is important to set appropriate test conditions for accurate screening. Furthermore, the freezing tolerance as a practical baker's yeast can be evaluated by carrying out more accurate and practical dough-based fermentation power evaluation without being limited to liquid system evaluation.
実用パン酵母の生地系での冷凍耐性は、ファーモグラフII（ＡＴＴＯ）を用い、冷凍前および解凍後の生地からのガス発生量により評価できる。 The freezing tolerance in the dough system of practical baker's yeast can be evaluated by the amount of gas generated from the dough before freezing and after thawing using Farmograph II (ATTO).
（実用株へのウラシル要求性の賦与）
実用株（実用パン酵母）の形質転換を実施するに当たり、遺伝子導入の成否を判定するための選択マーカーが必要となる。実用パン酵母にウラシル要求性を賦与するには、菌体を５−ＦＯＡ（５-フルオロオロチン酸）寒天培地に塗布し、３０℃で培養し、生育したコロニーを選抜すればよい。
(Granting uracil requirements to practical strains)
In carrying out transformation of a practical strain (practical baker's yeast), a selection marker for determining the success or failure of gene transfer is required. In order to impart uracil requirement to practical baker's yeast, the bacterial cells may be applied to 5-FOA (5-fluoroorotic acid) agar medium, cultured at 30 ° C., and grown colonies may be selected.
（遺伝子高発現株の作製）
ＫＴＲ１遺伝子及びＭＮＴ３遺伝子の少なくとも一つを高発現させるには、該遺伝子を含む多コピー型プラスミドで酵母を形質転換するか、該遺伝子を酵母で効率良く機能するプロモーター配列に連結して得られる組換えＤＮＡを用いて酵母を形質転換するか、あるいは酵母で効率良く機能する他のプロモーター配列を、染色体ＤＮＡ上の該遺伝子のプロモーター配列と置換、或いは該遺伝子の上流側に挿入すればよい。また、ＫＴＲ１及び／又はＭＮＴ３遺伝子をより活性の高いものと置換してもよい。
(Production of high gene expression strain)
To highly express at least one of the KTR1 gene and the MNT3 gene, a combination obtained by transforming yeast with a multicopy plasmid containing the gene or ligating the gene to a promoter sequence that functions efficiently in yeast. The transformed DNA may be used to transform yeast, or another promoter sequence that functions efficiently in yeast may be replaced with the promoter sequence of the gene on the chromosomal DNA, or inserted upstream of the gene. Alternatively, the KTR1 and / or MNT3 gene may be replaced with a more active one.
（遺伝子発現量の規定と測定方法）
酵母では、各遺伝子の発現量は、様々なストレスを与えることにより変化することが知られている。例えば、浸透圧ストレス下では、細胞内の浸透圧を高めるために、グリセロール合成に関わる遺伝子の発現量が増加する。同様に、冷凍ストレス環境下では、冷凍ストレス緩和に関わる遺伝子の発現量が増加していると考えられる。従って、冷凍耐性遺伝子を高発現することにより、酵母の冷凍耐性を向上させることが可能であると推察される。ここで、本発明において遺伝子高発現とは、遺伝子発現量が元株の５倍以上、好ましくは１０倍以上に増加した株を高発現株と定義する。遺伝子の発現量の比較は、転写されたｍＲＮＡの量又は翻訳されたタンパク質の量を比較することにより行うことができる。ｍＲＮＡの検出を行うには、ノーザンブロット法、リアルタイムＰＣＲ等が用いられる。同様に、タンパク質の検出を行うには、ウェスタン解析を用いればよく、発現するタンパクに対する抗体を取得し、ＲＩもしくは蛍光ラベルを比較できる系の使用が望ましい。
(Regulation of gene expression level and measurement method)
In yeast, it is known that the expression level of each gene is changed by applying various stresses. For example, under osmotic stress, the expression level of genes involved in glycerol synthesis increases in order to increase intracellular osmotic pressure. Similarly, the expression level of genes involved in relieving freezing stress is considered to increase in a freezing stress environment. Therefore, it is presumed that the freezing tolerance of yeast can be improved by highly expressing the freezing tolerance gene. Here, in the present invention, high gene expression is defined as a high expression strain in which the gene expression level is increased to 5 times or more, preferably 10 times or more that of the original strain. Comparison of gene expression levels can be performed by comparing the amount of transcribed mRNA or the amount of translated protein. Northern blotting, real-time PCR, or the like is used to detect mRNA. Similarly, Western analysis may be used to detect a protein, and it is desirable to use a system capable of obtaining antibodies against expressed proteins and comparing RI or fluorescent labels.
以下に実施例を示し、本発明をより具体的に説明するにおいて、ＫＴＲ１遺伝子およびＭＮＴ３遺伝子が、パン酵母の冷凍耐性形質の賦与あるいは強化に関与する遺伝子である事を示すが、本発明は以下の実施例により何ら制限されない。 In the following, the present invention will be described in more detail with reference to examples, and it will be shown that the KTR1 gene and the MNT3 gene are genes involved in the impartation or enhancement of the freezing tolerance traits of baker's yeast. The embodiment is not limited at all.
本発明で用いた培地、プラスミド、ＤＮＡ、種々の酵素、大腸菌、酵母などの取り扱いに関する常法は、以下の参考文献に詳しく記載されており、本発明ではこれらに従い実施した。
参考文献１：「酵母遺伝子実験マニュアル」 大矢禎一監訳 丸善
参考文献２：「バイオ実験イラストレイテッド １ 分子生物学実験の基礎」 中山広樹、西方敬人著 秀潤社
参考文献３：「バイオ実験イラストレイテッド ２ 遺伝子解析の基礎」 中山広樹、西方敬人著 秀潤社
参考文献４：「バイオ実験イラストレイテッド ３ 本当にふえるＰＣＲ」 中山広樹著 秀潤社
参考文献５：「バイオマニュアルシリーズ１０ 酵母による遺伝子実験法」 山本正幸編、羊土社
Conventional methods relating to the handling of the medium, plasmid, DNA, various enzymes, E. coli, yeast, etc. used in the present invention are described in detail in the following references, and were carried out according to these in the present invention.
Reference 1: “Yeast Genetic Experiment Manual” Translated by Junichi Oya Maruzen Reference 2: “Bio-Experiment Illustrated 1 Basics of Molecular Biology Experiment” Hiroki Nakayama, Takato Nishikata Shujunsha Reference 3: “Bio-Experiment Illustration” "Rated 2 Gene Analysis Fundamentals" Hiroki Nakayama, Takato Nishikata, Shujunsha Reference 4: "
（実施例１） 遺伝子破壊株ライブラリーからの冷凍耐性遺伝子スクリーニング
遺伝子破壊株ライブラリーを、ＹＰＤ培地を用いてマイクロプレートで静置培養し、培養液をドライアイス・エタノールバスにより５分間冷却後、−８０℃の冷凍庫で１日冷凍保存した。冷凍前後の培養液を適宜希釈して、それぞれＹＰＤ寒天培地にスポットし、増殖の様子を目視することにより、冷凍後の増殖が著しく低下した菌株を冷凍感受性株として一次スクリーニングした。一次スクリーニングした遺伝子破壊株については、改めて二次スクリーニングで冷凍後の生存率を測定した。ＹＰＤ培地５ｍｌに適量の菌体を植菌して３０℃で一晩前培養した後、培養液５０μｌを新しいＹＰＤ培地５ｍｌに植菌し、３０℃で一晩本培養した。培養液を１ｍｌずつ１．５ｍｌマイクロチューブ２本に分注し、１本は滅菌水で適宜希釈して、ＹＰＤ寒天培地に塗布した。もう１本は−８０℃のドライアイス・エタノールバスで５分間冷却後、−８０℃の冷凍庫で冷凍保存した。１日冷凍後、２５℃で１５分間静置して解凍した培養液サンプルを、滅菌水で適宜希釈して、ＹＰＤ寒天培地に塗布した。冷凍前及び冷凍後のサンプルを塗布した寒天培地に生育したコロニーをカウントし、冷凍後の菌体生存率（冷凍後サンプルのコロニー数／冷凍前サンプルのコロニー数）を指標として冷凍耐性の評価を行った。図１に示すように、ＹＯＲ０９９Ｗ、ＹＩＬ０１４Ｗ、ＹＢＬ０９０Ｗ、ＹＫＬ１２６Ｗ、ＹＤＲ１１９Ｗの何れか一つの遺伝子の破壊株は非破壊株と比べて冷凍後の生存率が２０％以上低下していた。このことから、これら５つの遺伝子は、冷凍耐性に関連していることが示唆された。
(Example 1) Freezing tolerance gene screening from gene disrupted strain library The gene disrupted strain library was statically cultured on a microplate using YPD medium, and the culture solution was cooled in a dry ice / ethanol bath for 5 minutes, Refrigerated at -80 ° C for 1 day. The culture broth before and after freezing was appropriately diluted, each spotted on a YPD agar medium, and the state of growth was visually observed, so that the strains with markedly reduced growth after freezing were primarily screened as frozen sensitive strains. For the gene-disrupted strains subjected to the primary screening, the survival rate after freezing was measured again in the secondary screening. An appropriate amount of cells were inoculated into 5 ml of YPD medium and pre-cultured overnight at 30 ° C., and then 50 μl of the culture solution was inoculated into 5 ml of new YPD medium, followed by main culture at 30 ° C. overnight. The culture solution was dispensed 1 ml at a time into two 1.5 ml microtubes, one was appropriately diluted with sterilized water, and applied to a YPD agar medium. The other was cooled in a −80 ° C. dry ice / ethanol bath for 5 minutes and then stored frozen in a −80 ° C. freezer. After freezing for 1 day, the culture solution sample which was allowed to stand at 25 ° C. for 15 minutes and thawed was appropriately diluted with sterilized water and applied to a YPD agar medium. Count colonies grown on an agar medium coated with samples before and after freezing, and evaluate freezing resistance using the survival rate of cells after freezing (number of colonies after freezing / number of colonies before freezing) as an index. went. As shown in FIG. 1, the disrupted strain of any one gene of YOR099W, YIL014W, YBL090W, YKL126W, and YDR119W had a survival rate of 20% or more lower than that of the non-destructed strain. This suggested that these five genes are related to freezing tolerance.
このうち、タンパク質への糖鎖付加に関わる遺伝子として報告があるＫＴＲ１遺伝子（ＹＯＲ０９９Ｗ）とＭＮＴ３遺伝子（ＹＩＬ０１４Ｗ）の両遺伝子については、新たな機能性として非常に興味深く、これらを詳細解析すると共に遺伝子高発現効果をパン生地系で評価することとした。 Of these, the KTR1 gene (YOR099W) and MNT3 gene (YIL014W), both of which have been reported as genes involved in glycosylation to proteins, are very interesting as new functionalities. The expression effect was evaluated using a bread dough system.
（実施例２） ＫＴＲ１遺伝子高発現プラスミドの作製
高発現プロモーターを含むプラスミドのプロモーター部分、例えば市販のプラスミドｐＡＵＲ１２３を制限酵素ＢａｍＨIで切断して得られるＡＤＨ１プロモーター及びＡＤＨ１ターミネーターの配列部分（ＰＡＤＨ１−ＴＡＤＨ１）約１．０ｋｂを、多コピー型のプラスミド、例えば市販のプラスミドｐＥＳＣ-ＵＲＡ（ＴＯＹＯＢＯ）を制限酵素ＰｖｕIIで切断して得られる配列約５．３ｋｂと連結し、多コピー型高発現プラスミドｐＥＵＡ約６．３ｋｂを作製した（図２）。
(Example 2) Preparation of KTR1 gene high-expression plasmid A promoter portion of a plasmid containing a high-expression promoter, for example, the ADH1 promoter obtained by cleaving the commercially available plasmid pAUR123 with the restriction enzyme BamHI and the sequence portion of the ADH1 terminator ( PADH1- T ADH1 ) of about 1.0 kb was ligated with a sequence of about 5.3 kb obtained by cleaving a multicopy plasmid, for example, the commercially available plasmid pESC-URA (TOYOBO) with the restriction enzyme PvuII, and the multicopy high expression plasmid pEUA About 6.3 kb was produced (FIG. 2).
遺伝子のクローニングは、ＰＣＲ（ポリメラーゼチェーンリアクション）を用いた方法が常法となっている。データベース菌株の染色体を鋳型とし、配列番号３、４に示したプライマーを用いたＰＣＲにより、配列番号１に示すサッカロミセス・セレビシエのＫＴＲ１遺伝子を取得した。ＫＴＲ１遺伝子の５’末端に付加したＫｐｎIと３’末端に付加したＳａｌIの両制限酵素切断部位の間約１．１ｋｂを、ｐＥＵＡの同制限酵素ＫｐｎI、ＳａｌI切断部位に挿入し、ＫＴＲ１遺伝子高発現プラスミドｐＥＵＡＫ１約７．４ｋｂを作製した（図３）。 For gene cloning, a method using PCR (polymerase chain reaction) has become a common method. The KTR1 gene of Saccharomyces cerevisiae shown in SEQ ID NO: 1 was obtained by PCR using the chromosome of the database strain as a template and the primers shown in SEQ ID NOs: 3 and 4. About 1.1 kb between both restriction enzyme cleavage sites of KpnI added to the 5 ′ end of KTR1 gene and SalI added to the 3 ′ end is inserted into the cleavage sites of the same restriction enzymes KpnI and SalI of pEUA, and the KTR1 gene is highly expressed. A plasmid pEUAK1 of about 7.4 kb was prepared (FIG. 3).
（実施例３） ＭＮＴ３遺伝子高発現プラスミドの作製
配列番号５、６に示したプライマーを用いたＰＣＲにより、配列番号２に示すサッカロミセス・セレビシエのＭＮＴ３遺伝子を取得し、ＭＮＴ３遺伝子の５’末端に付加したＫｐｎIと３’末端に付加したＳａｌIの両制限酵素切断部位の間約１．９ｋｂを、ｐＥＵＡの同制限酵素ＫｐｎI、ＳａｌI切断部位に挿入する以外は実施例２と同様にして、ＭＮＴ３遺伝子高発現プラスミドｐＥＵＡＭ１約８．２ｋｂを作製した（図４）。
(Example 3) Preparation of MNT3 gene high expression plasmid The MNT3 gene of Saccharomyces cerevisiae shown in SEQ ID NO: 2 was obtained by PCR using the primers shown in SEQ ID NOS: 5 and 6, and added to the 5 ′ end of the MNT3 gene. About 1.9 kb between both restriction enzyme cleavage sites of KpnI and SalI added to the 3 ′ end was inserted into the restriction enzyme KpnI and SalI cleavage sites of pEUA in the same manner as in Example 2, except that An expression plasmid pEUAM1 of about 8.2 kb was prepared (FIG. 4).
（実施例４）ウラシル要求性実用パン酵母の作製
ウラシル要求性実用パン酵母は、以下のようにして作製した。二倍体実用パン酵母をＹＰＤ寒天培地に塗布し、紫外線を３０秒間照射後、３０℃で１日培養した。生育した菌体をレプリカ法により５−ＦＯＡ寒天培地に移しとり、３０℃で３〜４日培養し、生育したコロニーを選抜することにより取得した。ウラシル要求性が賦与（ＵＲＡ３欠損変異）されたことは、ウラシル要求性を相補、すなわちＵＲＡ３を含有するプラスミドＹＣｐ５０を用いて形質転換することにより、ウラシルを含有しないカザミノ酸培地で生育できるようになることで確認した。また、要求性の相補株では、野生型の二倍体実用パン酵母と同等の増殖性、発酵力を持つことも確認した。
(Example 4) Production of uracil-required practical baker's yeast A uracil-required practical baker's yeast was produced as follows. A diploid practical baker's yeast was applied to a YPD agar medium, irradiated with ultraviolet rays for 30 seconds, and then cultured at 30 ° C. for 1 day. The grown cells were transferred to a 5-FOA agar medium by the replica method, cultured at 30 ° C. for 3 to 4 days, and the grown colonies were selected. The addition of uracil auxotrophy (URA3-deficient mutation) makes it possible to grow in a casamino acid medium that does not contain uracil by complementing uracil auxotrophy, that is, by transforming with URA3-containing plasmid YCp50. I confirmed that. It was also confirmed that the required complementary strains had the same growth and fermentability as wild-type diploid practical baker's yeast.
（実施例５） 実用パン酵母でのＫＴＲ１遺伝子高発現菌株の作製と該遺伝子の発現量測定
実施例２で作製したプラスミドと実施例４で作製した宿主を用いて、酢酸リチウム法に準ずる方法によりＫＴＲ１高発現プラスミド（ｐＥＵＡＫ１）で形質転換したＧＭＫＹ７９株と、非高発現プラスミド（ｐＥＵＡ）で形質転換したＧＭＫＹ７８株を作製した。
(Example 5) Production of KTR1 gene high expression strain in practical baker's yeast and measurement of expression level of the gene Using the plasmid prepared in Example 2 and the host prepared in Example 4, by a method according to the lithium acetate method GMKY79 strain transformed with KTR1 high expression plasmid (pEUAK1) and GMKY78 strain transformed with non-high expression plasmid (pEUA) were prepared.
プラスミドの導入の確認は、形質転換株からＤＮＡ（染色体及びプラスミド）を抽出し、これを鋳型として、配列番号７、８に示したプライマーを用いてＰＣＲを行い、ＰＣＲ産物のＤＮＡサイズを測定することにより確認した。 Confirmation of the introduction of the plasmid is performed by extracting DNA (chromosome and plasmid) from the transformant, using this as a template, performing PCR using the primers shown in SEQ ID NOs: 7 and 8, and measuring the DNA size of the PCR product. Was confirmed.
プラスミドの導入が確認できた遺伝子高発現実用パン酵母について、ＫＴＲ１遺伝子の発現量を測定した。菌体を表１の培地を用いて試験管で培養し、常法［ＲＮｅａｓｙ Ｍｉｎｉ（ＱＩＡＧＥＮ）プロトコール参照］に従い約５×１０７個の細胞からｔｏｔａｌ ＲＮＡを抽出した。 The expression level of the KTR1 gene was measured for the gene-expressing practical baker's yeast in which the introduction of the plasmid was confirmed. The bacterial cells were cultured in a test tube using the medium shown in Table 1, and total RNA was extracted from about 5 × 10 7 cells according to a conventional method [see RNeasy Mini (QIAGEN) protocol].
次に、常法［ライトサイクラーＲＮＡマスターＳＹＢＲ Ｇｒｅｅｎ（タカラバイオ）プロトコール参照］に従い、リアルタイムＰＣＲによりＫＴＲ１遺伝子のｍＲＮＡ量、すなわち遺伝子の発現量を測定した。ＫＴＲ１の検出には配列番号９、１０に示したプライマーを用いた。また、ハウスキーピング遺伝子としてＴＤＨ１を用い、検出には配列番号１１、１２に示したプライマーを用いた。 Next, the mRNA amount of the KTR1 gene, that is, the gene expression level was measured by real-time PCR according to a conventional method [see the light cycler RNA master SYBR Green (Takara Bio) protocol]. For the detection of KTR1, the primers shown in SEQ ID NOs: 9 and 10 were used. Further, TDH1 was used as a housekeeping gene, and the primers shown in SEQ ID NOs: 11 and 12 were used for detection.
その結果、図５−ａに示すように、ＫＴＲ１遺伝子の高発現株では、非高発現株と比べ、ＫＴＲ１遺伝子のｍＲＮＡ量が充分増加していた。 As a result, as shown in FIG. 5-a, the KTR1 gene mRNA level was sufficiently increased in the high expression strain of the KTR1 gene compared to the non-high expression strain.
（実施例６） 実用パン酵母でのＭＮＴ３遺伝子高発現菌株の作製と該遺伝子の発現量測定
実施例３で作製したプラスミドと実施例４で作製した宿主を用いて、酢酸リチウム法に準ずる方法によりＭＮＴ３高発現プラスミド（ｐＥＵＡＭ１）で形質転換したＧＭＫＹ８０株を作製した。プラスミドの導入の確認およびｔｏｔａｌ ＲＮＡの抽出は、実施例５と同様にして行った。
(Example 6) Production of MNT3 gene high expression strain in practical baker's yeast and measurement of expression level of the gene Using the plasmid prepared in Example 3 and the host prepared in Example 4, a method according to the lithium acetate method GMKY80 strain transformed with MNT3 high expression plasmid (pEUAM1) was prepared. Confirmation of introduction of the plasmid and extraction of total RNA were carried out in the same manner as in Example 5.
次に、ＭＮＴ３遺伝子のｍＲＮＡ量、すなわち遺伝子の発現量を測定については、ＭＮＴ３の検出に配列番号１３、１４に示したプライマーを用いる以外は、実施例５と同様にして行った。 Next, the amount of MNT3 gene mRNA, that is, the gene expression level was measured in the same manner as in Example 5 except that the primers shown in SEQ ID NOs: 13 and 14 were used for detection of MNT3.
その結果、図５−ｂに示すように、ＭＮＴ３遺伝子の高発現株では、非高発現株と比べ、ＭＮＴ３遺伝子のｍＲＮＡ量が充分量増加していた。 As a result, as shown in FIG. 5-b, the amount of MNT3 gene mRNA was sufficiently increased in the high expression strain of MNT3 gene compared to the non-high expression strain.
（実施例７） ＫＴＲ１遺伝子高発現実用パン酵母の冷凍耐性評価
実施例５で作製したＫＴＲ１遺伝子高発現実用パン酵母は、表１の培地を用いて坂口フラスコで培養した。得られたそれぞれの培養酵母を吸引ろ過して、水分含量約７０％の固体状にした。これを表２の配合でパン生地に加えて混捏した。
(Example 7) Evaluation of freezing tolerance of KTR1 gene high expression practical baker's yeast The KTR1 gene high expression practical baker's yeast prepared in Example 5 was cultured in a Sakaguchi flask using the medium shown in Table 1. Each of the obtained cultured yeasts was suction filtered to obtain a solid having a water content of about 70%. This was added to the bread dough with the formulation of Table 2 and kneaded.
混捏後、２０ｇに分割し、３０℃で６０分の前発酵をとった後、１つは３８℃で１２０分間のガス発生量（冷凍前）をファーモグラフIIで測定した。残りは−２０℃で冷凍貯蔵し、１，２週間後、生地を解凍（２５℃、３０分）した。解凍後の生地も冷凍前の生地と同様に、３８℃で１２０分間のガス発生量（冷凍１Ｗ、２Ｗ）をファーモグラフIIで測定した。その結果は、表３に示す。上記の培養酵母について、冷凍前と解凍後の生地からのガス発生量から、冷凍耐性度を算出した。その結果は、図６に示す。 After kneading, after dividing into 20 g and pre-fermenting at 30 ° C. for 60 minutes, one measured the gas generation amount (before freezing) at 38 ° C. for 120 minutes by Farmograph II. The rest was stored frozen at −20 ° C., and after 1, 2 weeks, the dough was thawed (25 ° C., 30 minutes). As with the dough before freezing, the amount of gas generated (freezing 1 W, 2 W) for 120 minutes at 38 ° C. was measured with a Pharmagraph II. The results are shown in Table 3. About said cultured yeast, the freezing tolerance degree was computed from the gas generation amount from the dough before freezing and after thawing | decompression. The result is shown in FIG.
（実施例８） ＭＮＴ３遺伝子高発現実用パン酵母の冷凍耐性評価
実施例６で作製したＭＮＴ３遺伝子高発現実用パン酵母を用いた以外は、実施例７と同様にして固体状の培養酵母を得、冷凍耐性度を算出した。その結果は、表３及び図６に示す。
(Example 8) Evaluation of freezing tolerance of MNT3 gene high expression practical baker's yeast Except that the MNT3 gene high expression practical baker's yeast prepared in Example 6 was used, solid culture yeast was obtained in the same manner as in Example 7, The degree of freezing tolerance was calculated. The results are shown in Table 3 and FIG.
以上の結果より、ＫＴＲ１遺伝子やＭＮＴ３遺伝子を高発現させることで冷凍耐性が向上し、より実用性の高いパン酵母を作製することができた。更には新規な冷凍耐性パン酵母の作製方法を提供することができた。 From the above results, freezing tolerance was improved by highly expressing the KTR1 gene and the MNT3 gene, and baker's yeast with higher practicality could be produced. Furthermore, a novel method for producing freeze-resistant baker's yeast could be provided.
加えて、ＫＴＲ１遺伝子やＭＮＴ３遺伝子が糖鎖付加に関連する遺伝子であることから、本発明の遺伝子以外にも糖鎖付加に関連する遺伝子が冷凍耐性に関与していることを示唆できたものと言える。 In addition, since the KTR1 gene and the MNT3 gene are genes related to glycosylation, it can be suggested that genes related to glycosylation other than the gene of the present invention are involved in freezing tolerance. I can say that.
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